Characterization and expression of cathepsin B2 in Fasciola gigantica

Fasciola gigantica cathepsin B belongs to a family of cysteine proteases which is involved in invasion of host tissues. In this study, the recombinant cathepsin B2 (rFgCatB2), synthesized in Pichia pastoris, showed enzymatic activity on a fluorometric substrate Z-Phe-Arg-AMC and gelatin. Furthermore, this recombinant enzyme could degrade IgG and type I collagen. Mouse antiserum against rFgCatB2 reacted with the native FgCatB2 in whole body (WB) extracts of metacercariae (MET), newly excysted juveniles (NEJ) and 2 week-old juveniles, but not in 3, 4 week-old juveniles and adult flukes. Immunolocalization showed the presence of cathepsin B2 only in the caecal epithelium of MET, NEJ and 2 week-old juveniles. Co-localization of FgCatB2 and a prominent antigen of NEJ, FgCatB3, revealed that these proteins were expressed at the same regions in the caecal epithelium. Anti-rFgCatB2 showed no cross reaction with the other parasites’ antigens by Western blotting. These findings suggest that CatB2 is expressed only in early stages of the parasite and may be involved in digestion of host connective tissues and evasion of the host immune system during their penetration and migration. Thus, CatB2 could be considered as an immunodiagnostic and vaccine candidate for fasciolosis.     

Porcine muscle sensory attributes associate with major changes in gene networks involving CAPZB, ANKRD1, and CTBP2

Principal component analysis of traits related to carcass and meat properties were combined with microarray expression data for the identification of functional networks of genes and biological processes taking place during the conversion of muscle to meat. Principal components (PCs) with high loadings of meat quality traits were derived from phenotypic data of 572 animals of a porcine crossbreed population. Microarray data of 74 M. longissimus dorsi samples were correlated with PC datasets. Lists of significantly correlated genes were analyzed for enrichment of functional annotation groups as defined in the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases as well as the Ingenuity Pathways Analysis library. Ubiquitination, phosphorylation, mitochondrion dysfunction, actin, integrin, platelet-derived growth factor, epidermal growth factor, vascular endothelial growth factor, and Ca signaling pathways are correlated with meat quality. Among the significantly trait-associated genes, CAPZB, ANKRD1, and CTBP2 are promoted as candidate genes for meat quality that provide a link between the highlighted pathways. Knowledge of the relevant biological processes and the differential expression of members of the pathway will provide tools that are predictive for traits related to meat quality and that may also be diagnostic for many muscle defects or damages including muscle atrophy, dystrophy, and hypoxia.     

Integrative analyses of Nervilia (Orchidaceae) section Linervia reveal further undescribed cryptic diversity in Thailand

The delimitation of cryptic species is necessary to accurately classify and appropriately conserve biodiversity. Integrative analyses can be incisive in detecting and circumscribing cryptic diversity, especially in species complexes whose members are delineated by minor or overlapping morphological variation. We adopt an integrative approach to assess species relationships and resolve species boundaries in the taxonomically difficult Nervilia adolphi/punctata species alliance of N. sect. Linervia, an Old World complex of reduced, one-flowered terrestrial orchids that is both species-rich and poorly known in tropical and warm temperate Asia. We sampled 12 of the 27 known species of the alliance in Asia, including all four species reported from Thailand and a further 20 plants collected in that country that could not be satisfactorily identified using morphology alone. Phylogenetic analyses using one nuclear (ITS) and two plastid (matK and trnL-F) markers confirmed both N. sect. Linervia and the alliance itself as monophyletic, and corroborated 11 of the 12 sampled species; N. punctata proved polyphyletic, with the Thai samples referred to this Indonesian species falling sister to the Himalayan N. mackinnonii. The 20 unidentified Thai samples formed three distinct, strongly supported clades. STACEY, a Bayesian coalescence approach to species delimitation, resolved the same three clusters, but provided evidence suggesting that one comprised two distinct sub-clades. Building on this genetic evidence, we identify subtle morphological differences and invoke a diagnosable species concept to circumscribe three previously unrecognized cryptic species from Thailand. This objective approach to species delimitation validates ostensibly minor morphological differences as a basis for differentiating species within the alliance, paving the way for a global analysis of species boundaries throughout the genus as a whole.     

Association study and expression analysis of porcine ESR1 as a candidate gene for boar fertility and sperm quality

Male fertility is impaired through the lack of ESR1 (Estrogen Receptor 1) but little is known about the ESR1 roles in boar spermatogenesis and fertility. Therefore, this research was aimed at investigating the association with sperm quality and boar fertility traits in a total of 300 boars both from purebred Pietrain and Pietrain × Hampshire crosses. A SNP in coding region of ESR1g.672C>T in exon 1 was associated with sperm motility (P<0.05) and plasma droplet rate (P<0.01) while the polymorphism in non-coding region of ESR1g.35756T>C in inton 1 was associated with non-return rate (P<0.05). Furthermore, to analyse the mRNA and protein expression of ESR1 in boar reproductive tissues, a total of six boars were divided into two groups [Group I (G-I) and Group II (G-II)], where G-I had relatively better sperm quality. ESR1 expression was higher in tissues collected from G-I boars than those of collected from G-II boars, and the difference in mRNA expression was significant (P<0.01) in head of epididymis. The ESR1 protein expression results from western blot coincided with the results of qRT-PCR. The ESR1 protein localization observed a strong staining in the cytoplasm of Sertoli cell in the testis, in the epithelial cells in head and tail of epididymis, in smooth muscle in tail of epididymis, and in the post acrosomal region and tail of the spermatozoa. These results will improve the understanding of the functions of the ESR1 in spermatogenesis within the reproductive tract and will shed light on ESR1 as a candidate in the selection of boar with good sperm quality and fertility.     

Mapping of quantitative trait loci for mycoplasma and tetanus antibodies and interferon-gamma in a porcine F2 Duroc × Pietrain resource population

The aim of the present study was to detect quantitative trait loci (QTL) for innate and adaptive immunity in pigs. For this purpose, a Duroc × Pietrain F₂ resource population (DUPI) with 319 offspring was used to map QTL for the immune traits blood antibodies and interferon-gamma using 122 microsatellites covering all autosomes. Antibodies response to Mycoplasma hyopneumoniae and tetanus toxoid vaccine and the interferon-gamma (IFNG) serum concentration were measured at three different time points and were used as phenotypes. The differences of antibodies and interferon concentration between different time points were also used for the linkage mapping. Line-cross and imprinting QTL analysis, including two-QTL, were performed using QTL Express. A total of 30 QTL (12, 6, and 12 for mycoplasma, tetanus antibody, and IFNG, respectively) were identified at the 5% chromosome-wide-level significant, of which 28 were detected by line-cross and 2 by imprinting model. In addition, two QTL were identified on chromosome 5 using the two-QTL approach where both loci were in repulsion phase. Most QTL were detected on pig chromosomes 2, 5, 11, and 18. Antibodies were increased over time and immune traits were found to be affected by sex, litter size, parity, and month of birth. The results demonstrated that antibody and IFNG concentration are influenced by multiple chromosomal areas. The flanking markers of the QTL identified for IFNG on SSC5 did incorporate the position of the porcine IFNG gene. The detected QTL will allow further research in these QTL regions for candidate genes and their utilization in selection to improve the immune response and disease resistance in pig.     

Association study and expression analysis of CD9 as candidate gene for boar sperm quality and fertility traits

Cluster-of-differentiation antigen 9 (CD9) gene expressed in the male germ line stem cells is crucial for sperm-egg fusion, and was therefore selected as candidate gene for boar semen quality. The association of CD9 with boar sperm quality and fertility trait was analyzed using a total of 340 boars both from purebred Pietrain and Pietrain×Hampshire crosses. A single nucleotide polymorphism (g.358A>T) in intron 6 was significantly associated with sperm motility (MOT) (P<0.001), plasma droplet rate (PDR) (P<0.001) and abnormal spermatozoa rate (ASR) (P<0.01). Boars were divided into two groups with group 1 (G-I) boars having a higher SCON and SMOT, lower SVOL (sperm volume) and group 2 (G-II) having a lower SCON and SMOT, higher SVOL. The mRNA and protein expression levels were evaluated in reproductive, non-reproductive tissues and spermatozoa from G-I and G-II animals by using quantitative real-time PCR and western blotting. When both reproductive and non-reproductive tissues were examined, highest mRNA was expressed in prostate gland, then in the body of the epididymis, vas deferens and tail of the epididymis. In case of reproductive tissues, CD9 expression was higher in tissues and spermatozoa collected from G-I boars than those collected from G-II boars. The mRNA expression was significantly different (P<0.05) in body of epididymis from G-I and G-II boars. The CD9 protein expression results from western blot were coincided with the results of qRT-PCR. Moreover, CD9 protein localization in Leydig cells, Sertoli cells, epithelial cells and spermatozoa was remarkable which indicated the important role of CD9 in spermatogenesis process. By using mRNA and protein expression profiles, it could be shown that CD9 plays a crucial role during sperm development, especially within the epididymis where the maturation of the sperm, a key process for the sperm quality and motility takes place. These results will improve the understanding of the functions of the CD9 in spermatogenesis within the reproductive tracts and will shed light on CD9 as a candidate gene in the selection of good sperm quality boars.     

Association and expression study of MMP3, TGFβ1 and COL10A1 as candidate genes for leg weakness-related traits in pigs

The present study was aimed to determine the association between metalloproteinase 3 (MMP3), transforming growth factor beta 1 (TGF??1) and collagen type X alpha I (COL10A1) gene polymorphisms with traits related to leg weakness in pigs. Three hundred Duroc?×?Pietrain cross breds (DuPi) and 299 pigs of a commercial population (CP) were used for the experiment. DuPi animals were examined for 10 different traits describing leg and feet structure, osteochondrosis (OC) scores and bone density status. Data of OC score at condylus medialis humeri, condylus medialis femoris and distal epiphysis ulna regions of CP were used for association analysis. Significant association (P?<?0.05) was found for MMP3 SNP (g.158 C>T) with OC at head of femur and bone mineral density in the DuPi population. Association (P?<?0.05) was found between SNP of TGF??1 (g.180 G>A) with rear leg score and the principle component denoting both OC and feet and leg scores in the DuPi population. No association was found between COL10A1 (g.72 C>T) and leg weakness related traits. The associations of SNPs with OC traits could not be confirmed in the commercial population. Expression analysis of the three candidate genes was performed to compare between healthy and OC. TGF??1 was found to be highly expressed (P?<?0.05) in the OC compared to healthy cartilages, but no significant different expressions were observed for MMP3 and COL10A1 genes. The present finding suggested that TGF??1 and MMP3 genes variants have an effect on some of the leg weakness related traits.     

Epistatic QTL pairs associated with meat quality and carcass composition traits in a porcine Duroc × Pietrain population

Background

Quantitative trait loci (QTL) analyses in pig have revealed numerous individual QTL affecting growth, carcass composition, reproduction and meat quality, indicating a complex genetic architecture. In general, statistical QTL models consider only additive and dominance effects and identification of epistatic effects in livestock is not yet widespread. The aim of this study was to identify and characterize epistatic effects between common and novel QTL regions for carcass composition and meat quality traits in pig.

Methods

Five hundred and eighty five F₂ pigs from a Duroc × Pietrain resource population were genotyped using 131 genetic markers (microsatellites and SNP) spread over the 18 pig autosomes. Phenotypic information for 26 carcass composition and meat quality traits was available for all F₂ animals. Linkage analysis was performed in a two-step procedure using a maximum likelihood approach implemented in the QxPak program.

Results

A number of interacting QTL was observed for different traits, leading to the identification of a variety of networks among chromosomal regions throughout the porcine genome. We distinguished 17 epistatic QTL pairs for carcass composition and 39 for meat quality traits. These interacting QTL pairs explained up to 8% of the phenotypic variance.

Conclusions

Our findings demonstrate the significance of epistasis in pigs. We have revealed evidence for epistatic relationships between different chromosomal regions, confirmed known QTL loci and connected regions reported in other studies. Considering interactions between loci allowed us to identify several novel QTL and trait-specific relationships of loci within and across chromosomes.